In this exploratory grant we will systematically develop the first total chemical synthesis of lasso peptides of native structure. Lasso peptides are ribosomally synthesized bacterial natural products that have a broad spectrum of biological activities as antimicrobial or antiviral agents, and as potent receptor antagonists. Lasso peptides isolated from nature have ~20 amino acids and have a unique folded structure that is essential for their stability and for full biological activity. This folded structure has a macrolactam ring between the N-terminal amino acid and the side chain carboxyl of a glutamic acid or aspartic acid residue; the C-terminal linear peptide segment is threaded through this ring. The challenge in total chemical synthesis of lasso peptides is forming this threaded structure. Although the biosynthesis of lasso peptides is quite well understood and can be used to engineer novel sequences, to date there has been no successful total chemical synthesis of a lasso peptide of native folded structure. Here we describe novel approaches to the synthesis of lasso peptides. The synthetic strategies draw on the broad range of cutting edge synthetic and analytical methods in this laboratory, and are prioritized in order of increasing synthetic complexity to provide for versatile implementation of alternatives if/when synthetic obstacles are encountered. The proposed routes are novel extensions of the thioester-mediated, amide-forming native chemical ligation methods developed in this laboratory, used in combination with our extensive expertise in peptide synthetic methods. The folded structures of our synthetic products will be unambiguously determined by established NMR and mass spectrometric methods, and by the racemic protein crystallography method recently pioneered in this laboratory for the determination of unknown Xray structures. All synthetic products will be screened against a panel of pathogenic bacteria. Successful total chemical synthesis of lasso peptides will enable the systematic dissection of the structure-function relationships in this important class of natura products and will enable the use of a much wider range of building blocks for the exploitation of the lasso peptide scaffold in the development of novel antibacterials.